1887

Abstract

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5′ RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

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2018-02-21
2019-10-18
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References

  1. Silo-Suh L, Suh SJ, Phibbs PV, Ohman DE. Adaptations of Pseudomonas aeruginosa to the cystic fibrosis lung environment can include deregulation of zwf, encoding glucose-6-phosphate dehydrogenase. J Bacteriol 2005; 187: 7561– 7568 [CrossRef] [PubMed]
    [Google Scholar]
  2. Meyer KC, Sharma A, Brown R, Weatherly M, Moya FR et al. Function and composition of pulmonary surfactant and surfactant-derived fatty acid profiles are altered in young adults with cystic fibrosis. Chest 2000; 118: 164– 174 [CrossRef] [PubMed]
    [Google Scholar]
  3. Palmer KL, Mashburn LM, Singh PK, Whiteley M. Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology. J Bacteriol 2005; 187: 5267– 5277 [CrossRef] [PubMed]
    [Google Scholar]
  4. Son MS, Matthews WJ, Kang Y, Nguyen DT, Hoang TT. In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect Immun 2007; 75: 5313– 5324 [CrossRef] [PubMed]
    [Google Scholar]
  5. Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR et al. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci USA 2006; 103: 8487– 8492 [CrossRef] [PubMed]
    [Google Scholar]
  6. Muñoz-Elías EJ, McKinney JD. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 2005; 11: 638– 644 [CrossRef] [PubMed]
    [Google Scholar]
  7. Reinscheid DJ, Eikmanns BJ, Sahm H. Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. J Bacteriol 1994; 176: 3474– 3483 [CrossRef] [PubMed]
    [Google Scholar]
  8. Vanderwinkel E, de Vlieghere M. Physiology and genetics of isocitritase and the malate synthases of Escherichia coli. Eur J Biochem 1968; 5: 81– 90 [PubMed] [Crossref]
    [Google Scholar]
  9. Dunn MF, Ramírez-Trujillo JA, Hernández-Lucas I. Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology 2009; 155: 3166– 3175 [CrossRef] [PubMed]
    [Google Scholar]
  10. Molina I, Pellicer MT, Badia J, Aguilar J, Baldoma L. Molecular characterization of Escherichia coli malate synthase G. Differentiation with the malate synthase A isoenzyme. Eur J Biochem 1994; 224: 541– 548 [CrossRef] [PubMed]
    [Google Scholar]
  11. Vanni P, Giachetti E, Pinzauti G, McFadden BA. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B 1990; 95: 431– 458 [CrossRef] [PubMed]
    [Google Scholar]
  12. Mckinney JD, Höner Zu Bentrup K, Muñoz-Elías EJ, Miczak A, Chen B et al. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 2000; 406: 735– 738 [CrossRef] [PubMed]
    [Google Scholar]
  13. Nandakumar M, Nathan C, Rhee KY. Isocitrate lyase mediates broad antibiotic tolerance in Mycobacterium tuberculosis. Nat Commun 2014; 5: 4306 [CrossRef] [PubMed]
    [Google Scholar]
  14. Shan Y, Lazinski D, Rowe S, Camilli A, Lewis K. Genetic basis of persister tolerance to aminoglycosides in Escherichia coli. MBio 2015; 6: e00078-15 [CrossRef] [PubMed]
    [Google Scholar]
  15. Andrews SC, Robinson AK, Rodríguez-Quiñones F. Bacterial iron homeostasis. FEMS Microbiol Rev 2003; 27: 215– 237 [CrossRef] [PubMed]
    [Google Scholar]
  16. Kim EJ, Sabra W, Zeng AP. Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1. Microbiology 2003; 149: 2627– 2634 [CrossRef] [PubMed]
    [Google Scholar]
  17. Meyer JM, Neely A, Stintzi A, Georges C, Holder IA. Pyoverdin is essential for virulence of Pseudomonas aeruginosa. Infect Immun 1996; 64: 518– 523 [PubMed]
    [Google Scholar]
  18. Kim J, Park C, Imlay JA, Park W. Lineage-specific SoxR-mediated regulation of an endoribonuclease protects non-enteric bacteria from redox-active compounds. J Biol Chem 2017; 292: 121– 133 [CrossRef] [PubMed]
    [Google Scholar]
  19. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem 1987; 160: 47– 56 [CrossRef] [PubMed]
    [Google Scholar]
  20. Imperi F, Tiburzi F, Visca P. Molecular basis of pyoverdine siderophore recycling in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2009; 106: 20440– 20445 [CrossRef] [PubMed]
    [Google Scholar]
  21. Petrat F, Rauen U, de Groot H. Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology 1999; 29: 1171– 1179 [CrossRef] [PubMed]
    [Google Scholar]
  22. Petrat F, de Groot H, Rauen U. Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. Arch Biochem Biophys 2000; 376: 74– 81 [CrossRef] [PubMed]
    [Google Scholar]
  23. Pellicer MT, Fernandez C, Badía J, Aguilar J, Lin EC et al. Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection. Characterization of the glc promoter. J Biol Chem 1999; 274: 1745– 1752 [PubMed] [Crossref]
    [Google Scholar]
  24. Heo A, Jang HJ, Sung JS, Park W. Global transcriptome and physiological responses of Acinetobacter oleivorans DR1 exposed to distinct classes of antibiotics. PLoS One 2014; 9: e110215 [CrossRef] [PubMed]
    [Google Scholar]
  25. Fourquez M, Devez A, Schaumann A, Guéneuguès A, Jouenne T et al. Effects of iron limitation on growth and carbon metabolism in oceanic and coastal heterotrophic bacteria. Limnol Oceanogr 2014; 59: 349– 360 [CrossRef]
    [Google Scholar]
  26. Beier S, Gálvez MJ, Molina V, Sarthou G, Quéroué F et al. The transcriptional regulation of the glyoxylate cycle in SAR11 in response to iron fertilization in the Southern Ocean. Environ Microbiol Rep 2015; 7: 427– 434 [CrossRef] [PubMed]
    [Google Scholar]
  27. Laporte DC, Thorsness PE, Koshland DE. Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment. J Biol Chem 1985; 260: 10563– 10568 [PubMed]
    [Google Scholar]
  28. Choi YW, Park SA, Lee HW, Lee NG. Alteration of growth-phase-dependent protein regulation by a fur mutation in Helicobacter pylori. FEMS Microbiol Lett 2009; 294: 102– 110 [CrossRef] [PubMed]
    [Google Scholar]
  29. Ahn S, Jung J, Jang IA, Madsen EL, Park W. Role of glyoxylate shunt in oxidative stress response. J Biol Chem 2016; 291: 11928– 11938 [CrossRef] [PubMed]
    [Google Scholar]
  30. Farr SB, Kogoma T. Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev 1991; 55: 561– 585 [PubMed]
    [Google Scholar]
  31. Anjem A, Varghese S, Imlay JA. Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli. Mol Microbiol 2009; 72: 844– 858 [CrossRef] [PubMed]
    [Google Scholar]
  32. Kehres DG, Janakiraman A, Slauch JM, Maguire ME. Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H2O2, Fe2+, and Mn2+. J Bacteriol 2002; 184: 3151– 3158 [CrossRef] [PubMed]
    [Google Scholar]
  33. Yamamoto K, Ishihama A. Two different modes of transcription repression of the Escherichia coli acetate operon by IclR. Mol Microbiol 2003; 47: 183– 194 [CrossRef] [PubMed]
    [Google Scholar]
  34. Gui L, Sunnarborg A, Laporte DC. Regulated expression of a repressor protein: FadR activates iclR. J Bacteriol 1996; 178: 4704– 4709 [CrossRef] [PubMed]
    [Google Scholar]
  35. Maloy SR, Bohlander M, Nunn WD. Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation. J Bacteriol 1980; 143: 720– 725 [PubMed]
    [Google Scholar]
  36. Maloy SR, Nunn WD. Role of gene fadR in Escherichia coli acetate metabolism. J Bacteriol 1981; 148: 83– 90 [PubMed]
    [Google Scholar]
  37. Resnik E, Pan B, Ramani N, Freundlich M, Laporte DC. Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression. J Bacteriol 1996; 178: 2715– 2717 [CrossRef] [PubMed]
    [Google Scholar]
  38. Prost JF, Nègre D, Oudot C, Murakami K, Ishihama A et al. Cra-dependent transcriptional activation of the icd gene of Escherichia coli. J Bacteriol 1999; 181: 893– 898 [PubMed]
    [Google Scholar]
  39. Zhu LW, Xia ST, Wei LN, Li HM, Yuan ZP et al. Enhancing succinic acid biosynthesis in Escherichia coli by engineering its global transcription factor, catabolite repressor/activator (Cra). Sci Rep 2016; 6: 36526 [CrossRef] [PubMed]
    [Google Scholar]
  40. Ducey TF, Jackson L, Orvis J, Dyer DW. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae. Microb Pathog 2009; 46: 166– 170 [CrossRef] [PubMed]
    [Google Scholar]
  41. Massé E, Gottesman S. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 2002; 99: 4620– 4625 [CrossRef] [PubMed]
    [Google Scholar]
  42. Mellin JR, Goswami S, Grogan S, Tjaden B, Genco CA. A novel fur- and iron-regulated small RNA, NrrF, is required for indirect fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis. J Bacteriol 2007; 189: 3686– 3694 [CrossRef] [PubMed]
    [Google Scholar]
  43. Davis BM, Quinones M, Pratt J, Ding Y, Waldor MK. Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae. J Bacteriol 2005; 187: 4005– 4014 [CrossRef] [PubMed]
    [Google Scholar]
  44. Wilderman PJ, Sowa NA, Fitzgerald DJ, Fitzgerald PC, Gottesman S et al. Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci USA 2004; 101: 9792– 9797 [CrossRef] [PubMed]
    [Google Scholar]
  45. Vasil ML. How we learnt about iron acquisition in Pseudomonas aeruginosa: a series of very fortunate events. Biometals 2007; 20: 587– 601 [CrossRef] [PubMed]
    [Google Scholar]
  46. Zhang YF, Han K, Chandler CE, Tjaden B, Ernst RK et al. Probing the sRNA regulatory landscape of P. aeruginosa: post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility. Mol Microbiol 2017; 106: 919– 937 [CrossRef] [PubMed]
    [Google Scholar]
  47. Oglesby AG, Farrow JM, Lee JH, Tomaras AP, Greenberg EP et al. The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing. J Biol Chem 2008; 283: 15558– 15567 [CrossRef] [PubMed]
    [Google Scholar]
  48. Friedman DB, Stauff DL, Pishchany G, Whitwell CW, Torres VJ et al. Staphylococcus aureus redirects central metabolism to increase iron availability. PLoS Pathog 2006; 2: e87 [CrossRef] [PubMed]
    [Google Scholar]
  49. Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML. GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 2002; 45: 1277– 1287 [CrossRef] [PubMed]
    [Google Scholar]
  50. Kawakami T, Kuroki M, Ishii M, Igarashi Y, Arai H. Differential expression of multiple terminal oxidases for aerobic respiration in Pseudomonas aeruginosa. Environ Microbiol 2010; 12: 1399– 1412 [CrossRef] [PubMed]
    [Google Scholar]
  51. Park SJ, Gunsalus RP. Oxygen, iron, carbon, and superoxide control of the fumarase fumA and fumC genes of Escherichia coli: role of the arcA, fnr, and soxR gene products. J Bacteriol 1995; 177: 6255– 6262 [CrossRef] [PubMed]
    [Google Scholar]
  52. Tseng CP. Regulation of fumarase (fumB) gene expression in Escherichia coli in response to oxygen, iron and heme availability: role of the arcA, fur, and hemA gene products. FEMS Microbiol Lett 1997; 157: 67– 72 [CrossRef] [PubMed]
    [Google Scholar]
  53. Hassett DJ, Howell ML, Sokol PA, Vasil ML, Dean GE. Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native fumC. J Bacteriol 1997; 179: 1442– 1451 [CrossRef] [PubMed]
    [Google Scholar]
  54. Tortell PD, Maldonado MT, Price NM. The role of heterotrophic bacteria in iron-limited ocean ecosystems. Nature 1996; 383: 330– 332 [CrossRef]
    [Google Scholar]
  55. Tortell PD, Maldonado MT, Granger J, Price NM. Marine bacteria and biogeochemical cycling of iron in the oceans. FEMS Microbiol Ecol 1999; 29: 1– 11 [CrossRef]
    [Google Scholar]
  56. Arai H. Regulation and function of versatile aerobic and anaerobic respiratory metabolism in Pseudomonas aeruginosa. Front Microbiol 2011; 2: 103 [CrossRef] [PubMed]
    [Google Scholar]
  57. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000; 406: 959– 964 [CrossRef] [PubMed]
    [Google Scholar]
  58. Comolli JC, Donohue TJ. Differences in two Pseudomonas aeruginosa cbb 3 cytochrome oxidases. Mol Microbiol 2004; 51: 1193– 1203 [CrossRef] [PubMed]
    [Google Scholar]
  59. Cunningham L, Williams HD. Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa. J Bacteriol 1995; 177: 432– 438 [CrossRef] [PubMed]
    [Google Scholar]
  60. Fujiwara T, Fukumori Y, Yamanaka T. A novel terminal oxidase, cytochrome baa 3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state. J Biochem 1992; 112: 290– 298 [CrossRef] [PubMed]
    [Google Scholar]
  61. Alvarez-Ortega C, Harwood CS. Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol 2007; 65: 153– 165 [CrossRef] [PubMed]
    [Google Scholar]
  62. Meylan S, Porter CBM, Yang JH, Belenky P, Gutierrez A et al. Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control. Cell Chem Biol 2017; 24: 195– 206 [CrossRef] [PubMed]
    [Google Scholar]
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